Microbial contamination by, for example, Gram positive bacteria, Gram negative bacteria, yeast, fungi, and molds may cause severe illness and, in some cases, even death in humans. Manufacturers in certain industries, for example, the pharmaceutical, medical device, and food industries, must meet exacting standards to verify that their products do not contain levels of microbial contaminants that would otherwise compromise the health of the recipient. These industries require frequent, accurate, and sensitive testing for the presence of such microbial contaminants to meet certain standards, for example, standards imposed by the United States Food and Drug Administration (USFDA) or Environmental Protection Agency. By way of example, the USFDA requires certain manufacturers of pharmaceuticals and invasive medical devices to establish that their products are free of detectable levels of Gram negative bacterial endotoxin.
Furthermore, when people become infected with Gram negative bacteria, the bacteria may produce and secrete fever-inducing bacterial endotoxins. Bacterial endotoxins can be dangerous and even deadly to humans. Symptoms of infection may range from fever, in mild cases, to death. In order to promptly initiate proper medical treatment, it usually is important to identify, as early as possible, the presence of an endotoxin and, if possible, the concentration of the endotoxin in the patient.
To date, a variety of assays have been developed to detect the presence and/or amount of a microbial contaminant in a test sample. One family of assays use hemocyte lysates prepared from the hemolymph of crustaceans, for example, horseshoe crabs. These assays typically exploit, in one way or another, a clotting cascade that occurs when the hemocyte lysate is exposed to a microbial contaminant. A currently preferred hemocyte lysate is amebocyte lysate (AL) produced from the hemolymph of a horseshoe crab, for example, Limulus polyphemus, Tachypleus gigas, Tachypleus tridentatus, and Carcinoscorpius rotundicauda. Amebocyte lysates produced from the hemolymph of Limulus, Tachypleus, and Carcinoscorpius species are referred to as Limulus amebocyte lysate (LAL), Tachypleus amebocyte lysate (TAL), and Carcinoscorpius amebocyte lysate (CAL), respectively.
Routine assays that use LAL include, for example, gel clot assays, end point turbidometric assays, kinetic turbidometric assays, and endpoint chromogenic assays (Prior (1990) “Clinical Applications of the Limulus Amebocyte Lysate Test” CRC PRESS 28-34). These assays, however, suffer from one or more disadvantages including reagent expense, assay speed and limited sensitivity ranges. Also, these assays typically require that samples be sent to a testing facility removed from the origin of the sample being tested. As a result, it may take hours to weeks before a problem can be detected and remedied. Accordingly, there is an ongoing need for faster and more sensitive methods, and portable test systems employing such methods, that overcome the need to submit samples to an off-site testing facility.